Cellulases or cellulytic enzymes are enzymes involved in hydrolyses of cellulose. In the hydrolysis of native cellulose, it is known that there are three major types of cellulase enzymes involved, namely cellobiohydrolase (1,4-.beta.-D-glucan cellobiohydrolase, EC 3.2.1.91), endo-.beta.-1,4-glucanase (endo-1,4-.beta.-D-glucan 4-glucanohydrolase, EC 3.2.1.4) and .beta.-glucosidase (EC 3.2.1.21).
Cellulases are synthesized by a large number of microorganisms which include fungi, actinomycetes, myxobacteria and true bacteria but also by plants. Especially endoglucanases of a wide variety of specificities have been identified.
A very important industrial use of cellulytic enzymes is the use for treatment of cellulosic textile or fabric, e.g. as ingredients in detergent compositions or fabric softener compositions, for bio-polishing of new fabric (garment finishing), and for obtaining a "stonewashed" look of cellulose-containing fabric, especially denim, and several methods for such treatment have been suggested, e.g. in GB-A-1 368 599, EP-A-0 307 564 and EP-A-0 435 876, WO 91/17243, WO 91/10732, WO 91/17244, PCT/DK95/000108 and PCT/DK95/00132.
Another important industrial use of cellulytic enzymes is the use for treatment of paper pulp, e.g. for improving the drainage or for deinking of recycled paper.
Especially the endoglucanases (EC No. 3.2.1.4) constitute an interesting group of hydrolases for the mentioned industrial uses. Endoglucanases catalyses endo hydrolysis of 1,4-.beta.-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxy methyl cellulose and hydroxy ethyl cellulose), lichenin, .beta.-1,4 bonds in mixed .beta.-1,3 glucans such as cereal .beta.-D-glucans or xyloglucans and other plant material containing cellulosic parts. The authorized name is endo-1,4-.beta.-D-glucan 4-glucano hydrolase, but the abbreviated term endoglucanase is used in the present specification. Reference can be made to T.-M. Enveri, "Microbial Cellulases" in W. M. Fogarty, Microbial Enzymes and Biotechnology, Applied Science Publishers, p. 183-224 (1983); Methods in Enzymology, (1988) Vol. 160, p. 200-391 (edited by Wood, W. A. and Kellogg, S. T.); Beguin, P., "Molecular Biology of Cellulose Degradation", Annu. Rev. Microbiol. (1990), Vol. 44, pp. 219-248; Beguin, P. and Aubert, J-P., "The biological degradation of cellulose", FEMS Microbiology Reviews 13 (1994) p.25-58; Henrissat, B., "Cellulases and their interaction with cellulose", Cellulose (1994), Vol. 1, pp. 169-196.
Fungal endoglucanases have been described in numerous publications, especially those derived from species as e.g. Fusarium oxysporum, Trichoderma reesei, Trichoderma longibrachiatum, Aspergillus aculeatus, Neocallimastix patriciarum, and e.g. from species of the genera Piromyces, Humicola, Myceliophthora, Geotricum, Penicillium, Irpex, Coprinus.
For example, fungal endoglucanases have been described by Sheppard, P. O., et al., "The use of conserved cellulase family-specific sequences to clone Cellulase homologue cDNAs from Fusarium oxysporum, Gene, (1994), Vol. 15, pp. 163-167, Saloheimo, A., et al., "A novel, small endoglucanase gene, egI5, from Trichoderma reesei isolated by expression in yeast", Molecular Microbiology (1994), Vol. 13(2), pp. 219-228; van Arsdell, J. N. et al., (1987), Cloning, characterization, and expression in Saccharomyces cerevisiae of endoglucanase I from Trichoderma reesei, Bio/Technology 5:60-64; Penttila, M. et al., (1986), "Homology between cellulase genes of Trichoderma reesei: complete nucleotide sequence of the endoglucanase I gene", Gene 45:253-263, Saloheimo, M. et al, (1988), "EGIII, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme", Gene 63:11-21; Gonzales, R., et al., "Cloning, sequence analysis and yeast expression of the egll gene from Trichoderma longibrachiatum", Appl. Microbiol. Biotechnol., (1992), Vol. 38, pp. 370-375; Ooi, T. et al. "Cloning and sequence analysis of a cDNA for cellulase (FI-CMCase) from Aspergillus aculeatus", Curr. Genet., (1990), Vol. 18, pp. 217-222; Ooi, T. et al, "Complete nucleotide sequence of a gene coding for Aspergillus aculeatus cellulase (FI-CMCase)", Nucleic Acids Research, (1990), Vol. 18, No. 19, p. 5884; Xue, G. et al., "Cloning and expression of multiple cellulase cDNAs from the anaerobic rumen fungus Neocallimastix patriciarum in E. coli", J. Gen. Microbiol., (1992), Vol. 138, pp. 1413-1420; Xue, G. et al., "A novel polysaccharide hydrolase cDNA (celD) from Neocallimastix patriciarum encoding three multi-functional catalytical domains with high endoglucanase, cellobiohydrolase and xylanase activities", J. Gen. Microbiol., (1992), Vol. 138, pp. 2397-2403; Zhou, L. et al., "Intronless celB from the anaerobic fungus Neocallimastix patriciarum encodes a modular family A endoglucanase", Biochem. J., (1994), Vol. 297, pp. 359-364; Dalb.o slashed.ge, H. and Heldt-Hansen, H. P., "A novel method for efficient expression cloning of fungal enzyme genes", Mol. Gen. Genet., (1994), Vol. 243, pp. 253-260; Ali, B. R. S. et al., "Cellulases and hemicellulases of the anaerobic fungus Piromyces constitute a multiprotein cellulose-binding complex and are encoded by multigene families", FEMS Microbiol. Lett., (1995), Vol. 125, No. 1, pp. 15-21. Further, the DNA Data Bank of Japan (DDBJ database publicly available at Internet) comprises two DNA sequences cloned from Penicillium janthinellum encoding endoglucanases (cloned by A. Koch and G. Mernitz, respectively) and a DNA sequence cloned from Humicola grisea var. thermoidea encoding an endoglucanase (cloned by T. Uozumi). Two endoglucanases from Macrophomina phaseolina have been cloned and sequenced, see Wang, H. Y. and Jones, R. W.: "Cloning, characterization and functional expression of an endoglucanase-encoding gene from the phytopathogenic fungus Macrophomina phaseolina" in Gene, 158:125-128, 1995, and Wang, H. Y. and Jones, R. W.: "A unique endoglucanase-encoding gene cloned from the phytopathogenic fungus Macrophomina phaseolina" in Applied And Environmental Microbiology, 61:2004-2006, 1995. One of these endoglucanases shows high homology to the egl3 endoglucanase from the fungus Trichoderma reesei, the other shows homology to the egl1 from the microbial phytopathogen Pseudomonas solanacearum indicating that both endoglucanases belong to family 5 of glycosyl hydrolases (B. Henrissat, Biochem J 280:309-316 (1991)). Filament-specific expression of a cellulase gene in the dimorphic fungus Ustilago maydis is disclosed in Schauwecker, F. et al. (1995).
WO 91/17243 (Novo Nordisk A/S) discloses a cellulase preparation consisting of a homogenous endoglucanase component immunoreactive with an antibody raised against a highly purified 43 kDa endoglucanase derived from Humicola insolens, DSM 1800; WO 91/17244 (Novo Nordisk A/S) discloses a new (hemi)cellulose degrading enzyme, such as an endoglucanase, a cellobiohydrolase or a .beta.-glucosidase, which may be derived from fungi other than Trichoderma and Phanerochaete; WO 93/20193 discloses an endoglucanase derivable from Aspergillus aculeatus; WO 94/21801 (Genencor Inc.) concerns a cellulase system isolated from Trichoderma longibrachiatum exhibiting endoglucanase activity; WO 94/26880 (Gist Brocades N. V.) discloses an isolated mixture of cellulose degrading enzymes, which preferable are obtained from Trichoderma, Aspergillus or Disporotrichum, comprising endoglucanase, cellobiohydrolase, and xyloglucanase activity; and WO 95/02043 (Novo Nordisk A/S) describes an enzyme with endoglucanase activity derived from Trichoderma harzianum, which can be used for a number of purposes including e.g. degradation or modification of plant cell walls.
It is also known that cellulases may or may not have a cellulose binding domain (a CBD). The CBD enhances the binding of the enzyme to a cellulose-containing fiber and increases the efficacy of the catalytic active part of the enzyme.
There is an ever existing need for providing novel cellulase enzyme preparations which may be used for applications where cellulase, preferably an endoglucanase, activity is desirable.
The object of the present invention is to provide novel enzyme preparations having substantial cellulytic activity at acid, neutral or alkaline conditions and improved performance in paper pulp processing, textile treatment, laundry processes or in animal feed; preferably novel cellulases, more preferably well-performing endoglucanases, which are contemplated to be producible or produced by recombinant techniques.